PROJECT SUMMARY The ideal scaffold to promote the scar-free repair of damaged tissues should utilize the basic elements that nature uses to assemble tissues in vivo. The architecture of the extracellular matrix (ECM) is not static during tissue formation and dynamically changes during development, repair and ageing. The tissue engineer's toolkit is limited by the lack of knowledge regarding relative ECM composition and turnover. The dearth of information on ECM composition and turnover rate can be attributed to the insoluble, interpenetrating networks into which the components are assembled. Researchers have begun to address this gap in knowledge by combining decellularization techniques, to enrich for ECM, with mass spectroscopic analysis of enzyme-digested peptide fragments. Together with recent advances protein labeling and quantitative proteomic analysis, these methods enable the identification and quantitation of proteins within the ECM. Bioorthogonal noncanonical amino acid labeling (BONCAT), utilizes cellular metabolism to incorporate amino acid analogs into newly synthesized proteins. The amino acid analogs carry small chemically functional groups (e.g. alkynes, azides) and can be labeled with affinity tags via click chemistry. These tags facilitate the enrichment of low abundance proteins that have incorporated the amino acid analogs, allowing for the direct comparison of protein synthesis in response to stimuli. Fluorescent non-canonical amino acid tagging (FUNCAT) introduces fluorescent tags to intact tissues via click chemistry to enable the direct visualization of protein distribution. BONCAT and FUNCAT have been used to characterize the synthesis of intracellular proteins; however, the integration of amino acid analogues into the ECM network has not been shown. The combination of musculoskeletal ECM mechanics and experimental biology expertise of the PI (Calve) with the chemical biology and biochemistry expertise of our collaborator, Prof. Tamara Kinzer-Ursem, will enable our team to successfully carry our proposed studies that utilize BONCAT and FUNCAT to metabolically label ECM synthesis and quantify the relative composition and turnover within in the murine model. The innovation of this proposal is the combination of novel 3D imaging techniques and mass spectroscopy with bioorthogonal labeling of murine tissues in vivo to obtain a 4D map of ECM remodeling in the limb. The knowledge and techniques resulting from these studies will be critical for future investigations into ECM remodeling during development and repair of specific musculoskeletal tissues (e.g. muscle and tendon) and the subsequent design of engineered therapies to restore functionality to these tissues when damaged.